RNAi tools for controlling viroid diseases

[EN] Viroids are small (250-400 nucleotides), single-stranded, circular RNAs without protein-coding capacity that infect a large number of ornamental and crop plant species, causing high economic losses worldwide. Strategies to control viroid diseases have included the use of naturally resistant cultivars in breeding programs, the superinfection exclusion with mild strains, the expression of ribonucleases, sense or antisense (catalytic) RNAs and, more recently, RNA interference (RNAi)-based tools. Here, I review the different RNAi strategies used to control vimid infections in plants, with particular focus on highly specific artificial small RNA (art-sRNA)-based tools such as artificial microRNAs and synthetic trans-acting small interfering RNAs. The advantages and future perspectives of art-sRNA-based RNAi for controlling viroid diseases are discussed.This work was supported by grants from Ministerio de Ciencia, Innovacion y Universidades (MCIU, Spain), Agencia Estatal de Investigacion (AEI, Spain) and Fondo Europeo de Desarrollo Regional (FEDER, European Union) [RTI2018-095118-A-100 and RYC-2017-21648 to A.C.].Carbonell, A. (2022). RNAi tools for controlling viroid diseases. Virus Research. 313:1-6. https://doi.org/10.1016/j.virusres.2022.1987291631

La biografía como metacine: el caso de Man on the Moon

Actas del Segundo Congreso Internacional de Historia y Cine organizado por el Instituto de Cultura y Tecnología Miguel de Unamuno y celebrado del 9 al 11 de septiembre de 2010 en la Universidad Carlos III de MadridEl género cinematográfico llamado biopic ha demostrado su capacidad para performar el modo de autoconocernos. Más que esto, los biopics devienen en una manera de ordenar los eventos que cobran sentido mediante la ayuda de cadenas causales. Sin embargo, siempre existe la duda de si todo lo que nos contamos forma parte de nuestra ilusión sobre lo que somos. A pesar de esta duda escéptica, esto no es un obstáculo para impedir las capacidades performativas del género y para recrear el self. El modo en que damos orden y una estructura a nuestros eventos o procesos mediante cadenas causales son, en sí mismas, una manera de recrearnos. Milos Forman ha demostrado mediante películas como Man on the Moon que el cine puede ser una doble exploración sobre el self –el de alguien concreto –y la forma en la que le damos una estructura a la narración cinematográfica.The cinematographic form called biopic had demonstrated its capabilities to serve to perform the way of self knowledge. Over than this, biopics became a way to put in order different events of someone that to make sense with the help of causal chains. However, always remains the doubt of whether the things we narrate to ourselves are a delusion of what we think we are. But this is not an obstacle to block the performative capacities of the biopic, and to create the self. The way in what we give an order and a structure to our events or process with causal chains are itself a way to recreate ourselves. Milos Forman has demonstrated with films like Man on the moon that cinema can do an exercise of self reckoning using the open tools of biopic. On this way, biopics can be a double exploration about the self –of someone self –and the way of give a structure to cinema storytelling.Publicad

La ucronía intencional como relato moral

Conferencias y Comunicaciones del primer Congreso Internacional de literatura fantástica y ciencia ficción, celebrado del 6 al 9 de mayo de 2008 en la Universidad Carlos III de Madri

Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants

ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO–sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO–sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO–RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing.This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C.Carbonell Olivares, A. (2017). Immunoprecipitation and High–Throughput Sequencing of ARGONAUTE–Bound Target RNAs from Plants. En Plant Argonaute Proteins: Methods and Protocols. Springer Link. 93-112. https://doi.org/10.1007/978-1-4939-7165-7_693112Carbonell A, Carrington JC (2015) Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol 27:111–117. doi: 10.1016/j.pbi.2015.06.013Fang X, Qi Y (2016) RNAi in plants: an Argonaute-centered view. Plant Cell 28(2):272–285. doi: 10.1105/tpc.15.00920Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13(7):350–358. doi: 10.1016/j.tplants.2008.04.007Mallory A, Vaucheret H (2010) Form, function, and regulation of ARGONAUTE proteins. Plant Cell 22(12):3879–3889. doi: 10.1105/tpc.110.080671Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133(1):128–141. doi: 10.1016/j.cell.2008.02.033Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon GJ, Qi Y (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133(1):116–127. doi: 10.1016/j.cell.2008.02.034Zhang X, Niu D, Carbonell A, Wang A, Lee A, Tun V, Wang Z, Carrington JC, Chang CE, Jin H (2014) ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis. Nat Commun 5:5468. doi: 10.1038/ncomms6468Zhu H, Hu F, Wang R, Zhou X, Sze SH, Liou LW, Barefoot A, Dickman M, Zhang X (2011) Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145(2):242–256. doi: 10.1016/j.cell.2011.03.024Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y (2008) The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol 49(4):493–500. doi: 10.1093/pcp/pcn043Cuperus JT, Carbonell A, Fahlgren N, Garcia-Ruiz H, Burke RT, Takeda A, Sullivan CM, Gilbert SD, Montgomery TA, Carrington JC (2010) Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nat Struct Mol Biol 17(8):997–1003. doi: 10.1038/nsmb.1866Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y (2009) Rice MicroRNA effector complexes and targets. Plant Cell 21(11):3421–3435. doi: 10.1105/tpc.109.070938Carbonell A, Fahlgren N, Garcia-Ruiz H, Gilbert KB, Montgomery TA, Nguyen T, Cuperus JT, Carrington JC (2012) Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24(9):3613–3629. doi: 10.1105/tpc.112.099945Gilbert KB, Fahlgren N, Kasschau KD, Chapman EJ, Carrington JC, Carbonell A (2014) Preparation of multiplexed small RNA libraries from plants. Bio Protoc 4(21):e1275Wang L, Si Y, Dedow LK, Shao Y, Liu P, Brutnell TP (2011) A low-cost library construction protocol and data analysis pipeline for Illumina-based strand-specific multiplex RNA-seq. PLoS One 6(10):e26426. doi: 10.1371/journal.pone.002642

Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants

[EN] Artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are highly specific 21-nucleotide small RNAs designed to recognize and silence complementary target RNAs. Art-sRNAs are extensively used in gene function studies or for improving crops, particularly to protect plants against viruses. Typically, antiviral art-sRNAs are computationally designed to target one or multiple sites in viral RNAs with high specificity, and art-sRNA constructs are generated and introduced into plants that are subsequently challenged with the target virus(es). Numerous studies have reported the successful application of art-sRNAs to induce resistance against a large number of RNA and DNA viruses in model and crop species. However, the application of art-sRNAs as an antiviral tool has limitations, such as the difficulty to predict the efficacy of a particular art-sRNA or the emergence of virus variants with mutated target sites escaping to art-sRNA-mediated degradation. Here, we review the different classes, features, and uses of art-sRNA-based tools to induce antiviral resistance in plants. We also provide strategies for the rational design of antiviral art-sRNAs and discuss the latest advances in developing art-sRNA-based methodologies for enhanced resistance to plant viruses.This research was funded by grants RYC-2017-21648 and RTI2018-095118-A-100 from the Ministerio de Ciencia, Innovacion y Universidades (MCIU, Spain), Agencia Estatal de Investigacion (AEI, Spain), and Fondo Europeo de Desarrollo Regional (FEDER, European Union) to AC.Cisneros, AE.; Carbonell, A. (2020). Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants. Plants. 9(6):1-16. https://doi.org/10.3390/plants9060669S1169

Antiviral roles of plant ARGONAUTES

[EN] ARGONAUTES (AGOs) are the effector proteins functioning in eukaryotic RNA silencing pathways. AGOs associate with small RNAs and are programmed to target complementary RNA or DNA. Plant viruses induce a potent and specific antiviral RNA silencing host response in which AGOs play a central role. Antiviral AGOs associate with virus-derived small RNAs to repress complementary viral RNAs or DNAs, or with endogenous small RNAs to regulate host gene expression and promote antiviral defense. Here, we review recent progress towards understanding the roles of plant AGOs in antiviral defense. We also discuss the strategies that viruses have evolved to modulate, attenuate or suppress AGO antiviral functions.We thank members of the Carrington lab for useful and crucial discussions, and apologize to those colleagues whose work could not be cited because of space and reference limitations. This work was supported by grants from the National Science Foundation (MCB-1231726 and MCB-1330562) and National Institutes of Health (AI043288) to James C Carrington, and from the European Commission (H2020-MSCA-IF-2014-655841) to Alberto Carbonell.Carbonell, A.; Carrington, JC. (2015). Antiviral roles of plant ARGONAUTES. Current Opinion in Plant Biology. 27:111-117. https://doi.org/10.1016/j.pbi.2015.06.013S11111727Meister, G. (2013). Argonaute proteins: functional insights and emerging roles. Nature Reviews Genetics, 14(7), 447-459. doi:10.1038/nrg3462Poulsen, C., Vaucheret, H., & Brodersen, P. (2013). Lessons on RNA Silencing Mechanisms in Plants from Eukaryotic Argonaute Structures. The Plant Cell, 25(1), 22-37. doi:10.1105/tpc.112.105643Martínez de Alba, A. E., Elvira-Matelot, E., & Vaucheret, H. (2013). Gene silencing in plants: A diversity of pathways. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1829(12), 1300-1308. doi:10.1016/j.bbagrm.2013.10.005Csorba, T., Kontra, L., & Burgyán, J. (2015). viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence. Virology, 479-480, 85-103. doi:10.1016/j.virol.2015.02.028Vaucheret, H. (2008). Plant ARGONAUTES. Trends in Plant Science, 13(7), 350-358. doi:10.1016/j.tplants.2008.04.007Morel, J.-B., Godon, C., Mourrain, P., Béclin, C., Boutet, S., Feuerbach, F., … Vaucheret, H. (2002). Fertile Hypomorphic ARGONAUTE (ago1) Mutants Impaired in Post-Transcriptional Gene Silencing and Virus Resistance. The Plant Cell, 14(3), 629-639. doi:10.1105/tpc.010358Qu, F., Ye, X., & Morris, T. J. (2008). Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proceedings of the National Academy of Sciences, 105(38), 14732-14737. doi:10.1073/pnas.0805760105Wang, X.-B., Jovel, J., Udomporn, P., Wang, Y., Wu, Q., Li, W.-X., … Ding, S.-W. (2011). The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes in Arabidopsis thaliana    . The Plant Cell, 23(4), 1625-1638. doi:10.1105/tpc.110.082305Dzianott, A., Sztuba-Solińska, J., & Bujarski, J. J. (2012). Mutations in the Antiviral RNAi Defense Pathway Modify Brome mosaic virus RNA Recombinant Profiles. Molecular Plant-Microbe Interactions®, 25(1), 97-106. doi:10.1094/mpmi-05-11-0137Garcia-Ruiz, H., Carbonell, A., Hoyer, J. S., Fahlgren, N., Gilbert, K. B., Takeda, A., … Carrington, J. C. (2015). Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection. PLOS Pathogens, 11(3), e1004755. doi:10.1371/journal.ppat.1004755Harvey, J. J. W., Lewsey, M. G., Patel, K., Westwood, J., Heimstädt, S., Carr, J. P., & Baulcombe, D. C. (2011). An Antiviral Defense Role of AGO2 in Plants. PLoS ONE, 6(1), e14639. doi:10.1371/journal.pone.0014639Jaubert, M., Bhattacharjee, S., Mello, A. F. S., Perry, K. L., & Moffett, P. (2011). ARGONAUTE2 Mediates RNA-Silencing Antiviral Defenses against Potato virus X in Arabidopsis    . Plant Physiology, 156(3), 1556-1564. doi:10.1104/pp.111.178012Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K. B., Montgomery, T. A., Nguyen, T., … Carrington, J. C. (2012). Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants  . The Plant Cell, 24(9), 3613-3629. doi:10.1105/tpc.112.099945Zhang, X., Zhang, X., Singh, J., Li, D., & Qu, F. (2012). Temperature-Dependent Survival of Turnip Crinkle Virus-Infected Arabidopsis Plants Relies on an RNA Silencing-Based Defense That Requires DCL2, AGO2, and HEN1. Journal of Virology, 86(12), 6847-6854. doi:10.1128/jvi.00497-12Ma, X., Nicole, M.-C., Meteignier, L.-V., Hong, N., Wang, G., & Moffett, P. (2014). Different roles for RNA silencing and RNA processing components in virus recovery and virus-induced gene silencing in plants. Journal of Experimental Botany, 66(3), 919-932. doi:10.1093/jxb/eru447Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M., & Watanabe, Y. (2008). The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins. Plant and Cell Physiology, 49(4), 493-500. doi:10.1093/pcp/pcn043Hamera, S., Song, X., Su, L., Chen, X., & Fang, R. (2011). Cucumber mosaic virus suppressor 2b binds to AGO4-related small RNAs and impairs AGO4 activities. The Plant Journal, 69(1), 104-115. doi:10.1111/j.1365-313x.2011.04774.xBhattacharjee, S., Zamora, A., Azhar, M. T., Sacco, M. A., Lambert, L. H., & Moffett, P. (2009). Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control. The Plant Journal, 58(6), 940-951. doi:10.1111/j.1365-313x.2009.03832.xRaja, P., Sanville, B. C., Buchmann, R. C., & Bisaro, D. M. (2008). Viral Genome Methylation as an Epigenetic Defense against Geminiviruses. Journal of Virology, 82(18), 8997-9007. doi:10.1128/jvi.00719-08Raja, P., Jackel, J. N., Li, S., Heard, I. M., & Bisaro, D. M. (2013). Arabidopsis Double-Stranded RNA Binding Protein DRB3 Participates in Methylation-Mediated Defense against Geminiviruses. Journal of Virology, 88(5), 2611-2622. doi:10.1128/jvi.02305-13Scholthof, H. B., Alvarado, V. Y., Vega-Arreguin, J. C., Ciomperlik, J., Odokonyero, D., Brosseau, C., … Moffett, P. (2011). Identification of an ARGONAUTE for Antiviral RNA Silencing in Nicotiana benthamiana        . Plant Physiology, 156(3), 1548-1555. doi:10.1104/pp.111.178764Ghoshal, B., & Sanfaçon, H. (2014). Temperature-dependent symptom recovery in Nicotiana benthamiana plants infected with tomato ringspot virus is associated with reduced translation of viral RNA2 and requires ARGONAUTE 1. Virology, 456-457, 188-197. doi:10.1016/j.virol.2014.03.026Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., Matsumoto-Yokoyama, E., Mitsuhara, I., … Ishikawa, M. (2010). In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90. Molecular Cell, 39(2), 282-291. doi:10.1016/j.molcel.2010.05.014Schuck, J., Gursinsky, T., Pantaleo, V., Burgyán, J., & Behrens, S.-E. (2013). AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system. Nucleic Acids Research, 41(9), 5090-5103. doi:10.1093/nar/gkt193Zhu, H., Duan, C.-G., Hou, W.-N., Du, Q.-S., Lv, D.-Q., Fang, R.-X., & Guo, H.-S. (2011). Satellite RNA-Derived Small Interfering RNA satsiR-12 Targeting the 3’ Untranslated Region of Cucumber Mosaic Virus Triggers Viral RNAs for Degradation. Journal of Virology, 85(24), 13384-13397. doi:10.1128/jvi.05806-11Cao, M., Du, P., Wang, X., Yu, Y.-Q., Qiu, Y.-H., Li, W., … Ding, S.-W. (2014). Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs inArabidopsis. Proceedings of the National Academy of Sciences, 111(40), 14613-14618. doi:10.1073/pnas.1407131111Smith, N. A., Eamens, A. L., & Wang, M.-B. (2011). Viral Small Interfering RNAs Target Host Genes to Mediate Disease Symptoms in Plants. PLoS Pathogens, 7(5), e1002022. doi:10.1371/journal.ppat.1002022Shimura, H., Pantaleo, V., Ishihara, T., Myojo, N., Inaba, J., Sueda, K., … Masuta, C. (2011). A Viral Satellite RNA Induces Yellow Symptoms on Tobacco by Targeting a Gene Involved in Chlorophyll Biosynthesis using the RNA Silencing Machinery. PLoS Pathogens, 7(5), e1002021. doi:10.1371/journal.ppat.1002021Navarro, B., Gisel, A., Rodio, M. E., Delgado, S., Flores, R., & Di Serio, F. (2012). Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing. The Plant Journal, 70(6), 991-1003. doi:10.1111/j.1365-313x.2012.04940.xMiozzi, L., Gambino, G., Burgyan, J., & Pantaleo, V. (2012). Genome-wide identification of viral and host transcripts targeted by viral siRNAs inVitis vinifera. Molecular Plant Pathology, 14(1), 30-43. doi:10.1111/j.1364-3703.2012.00828.xDe Ronde, D., Pasquier, A., Ying, S., Butterbach, P., Lohuis, D., & Kormelink, R. (2013). Analysis ofTomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression. Molecular Plant Pathology, 15(2), 185-195. doi:10.1111/mpp.12082Lacombe, S., Bangratz, M., Vignols, F., & Brugidou, C. (2010). The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing. The Plant Journal, 61(3), 371-382. doi:10.1111/j.1365-313x.2009.04062.xGuo, H., Song, X., Xie, C., Huo, Y., Zhang, F., Chen, X., … Fang, R. (2013). Rice yellow stunt rhabdovirus Protein 6 Suppresses Systemic RNA Silencing by Blocking RDR6-Mediated Secondary siRNA Synthesis. Molecular Plant-Microbe Interactions®, 26(8), 927-936. doi:10.1094/mpmi-02-13-0040-rOkano, Y., Senshu, H., Hashimoto, M., Neriya, Y., Netsu, O., Minato, N., … Namba, S. (2014). In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor    . The Plant Cell, 26(5), 2168-2183. doi:10.1105/tpc.113.120535Weinheimer, I., Jiu, Y., Rajamäki, M.-L., Matilainen, O., Kallijärvi, J., Cuellar, W. J., … Valkonen, J. P. T. (2015). Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants. PLOS Pathogens, 11(3), e1004711. doi:10.1371/journal.ppat.1004711Baumberger, N., Tsai, C.-H., Lie, M., Havecker, E., & Baulcombe, D. C. (2007). The Polerovirus Silencing Suppressor P0 Targets ARGONAUTE Proteins for Degradation. Current Biology, 17(18), 1609-1614. doi:10.1016/j.cub.2007.08.039Bortolamiol, D., Pazhouhandeh, M., Marrocco, K., Genschik, P., & Ziegler-Graff, V. (2007). The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing. Current Biology, 17(18), 1615-1621. doi:10.1016/j.cub.2007.07.061Csorba, T., Lózsa, R., Hutvágner, G., & Burgyán, J. (2010). Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1. The Plant Journal, 62(3), 463-472. doi:10.1111/j.1365-313x.2010.04163.xFusaro, A. F., Correa, R. L., Nakasugi, K., Jackson, C., Kawchuk, L., Vaslin, M. F. S., & Waterhouse, P. M. (2012). The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation. Virology, 426(2), 178-187. doi:10.1016/j.virol.2012.01.026Derrien, B., Baumberger, N., Schepetilnikov, M., Viotti, C., De Cillia, J., Ziegler-Graff, V., … Genschik, P. (2012). Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway. Proceedings of the National Academy of Sciences, 109(39), 15942-15946. doi:10.1073/pnas.1209487109Azevedo, J., Garcia, D., Pontier, D., Ohnesorge, S., Yu, A., Garcia, S., … Voinnet, O. (2010). Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes & Development, 24(9), 904-915. doi:10.1101/gad.1908710Zhang, X., Yuan, Y.-R., Pei, Y., Lin, S.-S., Tuschl, T., Patel, D. J., & Chua, N.-H. (2006). Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes & Development, 20(23), 3255-3268. doi:10.1101/gad.1495506Duan, C.-G., Fang, Y.-Y., Zhou, B.-J., Zhao, J.-H., Hou, W.-N., Zhu, H., … Guo, H.-S. (2012). Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein. The Plant Cell, 24(1), 259-274. doi:10.1105/tpc.111.092718Giner, A., Lakatos, L., García-Chapa, M., López-Moya, J. J., & Burgyán, J. (2010). Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs. PLoS Pathogens, 6(7), e1000996. doi:10.1371/journal.ppat.1000996Szabo, E. Z., Manczinger, M., Goblos, A., Kemeny, L., & Lakatos, L. (2012). Switching on RNA Silencing Suppressor Activity by Restoring Argonaute Binding to a Viral Protein. Journal of Virology, 86(15), 8324-8327. doi:10.1128/jvi.00627-12Pérez-Cañamás, M., & Hernández, C. (2015). Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing. Journal of Biological Chemistry, 290(5), 3106-3120. doi:10.1074/jbc.m114.593707Buchmann, R. C., Asad, S., Wolf, J. N., Mohannath, G., & Bisaro, D. M. (2009). Geminivirus AL2 and L2 Proteins Suppress Transcriptional Gene Silencing and Cause Genome-Wide Reductions in Cytosine Methylation. Journal of Virology, 83(10), 5005-5013. doi:10.1128/jvi.01771-08Soitamo, A. J., Jada, B., & Lehto, K. (2012). Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants. BMC Plant Biology, 12(1), 204. doi:10.1186/1471-2229-12-204Zhang, Z., Chen, H., Huang, X., Xia, R., Zhao, Q., Lai, J., … Xie, Q. (2011). BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis    . The Plant Cell, 23(1), 273-288. doi:10.1105/tpc.110.081695Várallyay, É., Válóczi, A., Ágyi, Á., Burgyán, J., & Havelda, Z. (2010). Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation. The EMBO Journal, 29(20), 3507-3519. doi:10.1038/emboj.2010.215Várallyay, É., & Havelda, Z. (2013). Unrelated viral suppressors of RNA silencing mediate the control of ARGONAUTE1 level. Molecular Plant Pathology, 14(6), 567-575. doi:10.1111/mpp.1202

Secondary Small Interfering RNA-Based Silencing Tools in Plants: An Update

Peer reviewe

Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance

[EN] RNA interference (RNAi)-based tools are used in multiple organisms to induce antiviral resistance through the sequence-specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi-based tools include artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). syn-tasiRNAs have emerged as a promising antiviral tool allowing for the multi-targeting of viral RNAs through the simultaneous expression of several syn-tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn-tasiRNA construct expressing four different syn-tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn-tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn-tasiRNA lines was not exclusive of lines with high syn-tasiRNA accumulation. Collectively, these results suggest that syn-tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn-tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn-tasiRNA.We thank V. Aragones and E. Moya for invaluable technical assistance. This work was supported by grants from Ministerio de Ciencia, Innovacion y Universidades (MCIU, Spain), Agencia Estatal de Investigacion (AEI, Spain) and Fondo Europeo de Desarrollo Regional (FEDER, European Union) (RTI2018-095118-A-100 and RYC-2017-21648 to A.C.; BIO2017-83184-R to J.-A.D.).Carbonell, A.; Lisón, P.; Daròs, J. (2019). Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance. 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Proving termination through conditional termination

We present a constraint-based method for proving conditional termination of integer programs. Building on this, we construct a framework to prove (unconditional) program termination using a powerful mechanism to combine conditional termination proofs. Our key insight is that a conditional termination proof shows termination for a subset of program execution states which do not need to be considered in the remaining analysis. This facilitates more effective termination as well as non-termination analyses, and allows handling loops with different execution phases naturally. Moreover, our method can deal with sequences of loops compositionally. In an empirical evaluation, we show that our implementation VeryMax outperforms state-of-the-art tools on a range of standard benchmarks.Peer ReviewedPostprint (author's final draft